JP6654323B2 - Cells capable of forming stratified epithelial tissue and method for producing the same - Google Patents

Description

  The present invention relates to a cell capable of forming a stratified epithelial tissue derived from a somatic cell, and a method for producing the same. Further, the present invention provides a cell preparation for regeneration of skin, mucous membrane or corneal tissue, a method for treating skin, mucous membrane or corneal ulcer, and a test substance for skin, mucous membrane or corneal disease, using the cell having the ability to form stratified epithelial tissue. The present invention relates to a method for judging the medicinal effect and a method for judging a stress caused by an external factor to the skin, mucous membrane or cornea. Furthermore, the present invention relates to a composition for preparing stratified epithelial cells for inducing somatic cells into stratified epithelial cells.

  The stratified epithelium plays the role of the outer skin, protecting the inside of the body from external factors such as mechanical injuries and infections in areas that come into contact with the outside world, such as the epidermis, mucous membranes and cornea, and at the same time, preventing the leakage and evaporation of blood and body fluids. ing. The stratified epithelial tissue is stratified and formed by self-renewal and differentiation of progenitor cells or stem cells existing in the basal layer.

  The most common conditions in which the integument is impaired are burns, trauma, iatrogenic damage (such as after tumor resection), external factors such as pressure ulcers, and skin caused by internal factors such as diabetes and peripheral circulatory failure. There is an ulcer. Any factor that causes dermatological damage can cause ulcers. Gastrointestinal ulcers in which the digestive tract outer skin is impaired, such as defects in the oral mucosa and anal mucosa, and corneal ulcers in which the cornea is impaired, are also included in the pathological conditions. Skin ulcers cause pain, infection, bleeding, etc. and, in widespread cases, are fatal by themselves. Mucosal ulcers can cause pain, infection, bleeding and gastrointestinal perforation. Corneal ulcers cause visual impairment. Due to the development of medicine and the spread of lifestyle-related diseases, it is expected that the number of people suffering from intractable skin ulcers due to pressure ulcers, diabetes ulcers, and blood circulation disorders will increase more and more in the future.

  Since epithelial cells have the properties of precursor cells and stem cells, ulcerative lesions of the epithelium are usually repaired by migration of epithelial cells existing around the ulcer. Therefore, in the case of a localized ulcerative lesion, scar healing can be obtained by keeping the wound clean and protecting it with an ointment or a wound dressing. However, ulcers are refractory when the wound is extensive, or when there are factors that inhibit healing, such as infection, malnutrition, hyperglycemia, or peripheral circulatory disorders. Conventionally, in such a case, treatment is performed by transplanting the skin from another part of the body through a surgical procedure such as skin grafting or flap plastic surgery. However, in particular, many patients having pressure ulcers, diabetic ulcers, and ulcers due to circulatory insufficiency have a poor general condition, and the risk associated with general anesthesia and surgical operation is high (see Non-Patent Documents 1 and 2). Also, when the wound is extremely extensive, such as in severe burns, the remaining self skin tissue alone may not be enough to reduce or close the ulcer area, and death may occur from infection or circulatory dysfunction. . In addition, flap surgery is required for the mucosal defect of the digestive tract, particularly the oral cavity, tongue, pharynx and anus, which is composed of stratified epithelium. In severe cases, corneal ulcers may require corneal transplantation.

  Therefore, in recent years, particularly for severe burns, a treatment for collecting a part of the autologous skin tissue, culturing the cell, and transplanting the autologous epidermal cell sheet created has been performed. However, patients who are eligible for this treatment lack the absolute amount of residual skin tissue, so the amount of skin tissue that can be used for collection and preparation of epidermal cell sheets is also insufficient, and there is no skin transplantation. However, it has often been difficult to create a sufficient amount of epidermal cell sheets in a few weeks during which the patient's survival can be obtained.

  In addition, for intractable ulcers such as pressure ulcers, diabetic ulcers, and ulcers due to circulatory failure, it may not always be possible to obtain closure of the wound by conventional surgical procedures such as skin grafting and flap plastic surgery . Surgical procedures, such as skin grafts and flap replacements, require the removal of the skin to be transplanted, which only places a strain on the body if the wound cannot be closed Therefore, a means for efficiently obtaining a wound epithelium in a less invasive manner was required.

  In recent years, a technique has been reported in which each gene encoding Oct3 / 4, Klf4, c-Myc, and Sox2 is introduced into somatic cells to initialize somatic cells and to induce induced pluripotent stem (iPS) cells. In addition, innovative technologies are provided in the field of regenerative medicine (see Patent Document 1, Non-Patent Document 3-4). However, since the production of iPS cells and the induction of iPS cells to epithelial cells have a long period of time, it is necessary to solve further technical problems in using this technique for severe burn patients. In addition, since the somatic cells acquire pluripotency during the process of iPS production, they are re-induced to epithelial cells.Therefore, there are various problems to solve the pluripotent state such as the possibility of canceration. In addition, because of poor production efficiency, further technical problems need to be solved for use in patients with intractable skin ulcers, mucosal ulcers, corneal ulcers and the like whose progress is relatively slow.

  On the other hand, in the process of development, somatic cells whose cell lineage has been determined continue to proliferate and differentiate within the same cell lineage. On the other hand, the conversion of a cell lineage that is not recognized physiologically is called direct conversion, and it is called skin conversion from skin fibroblasts to muscle cells (Non-patent Document 3), nerve cells (Non-patent Document 6), and cardiomyocytes (Non-patent Documents). 7), hepatocytes (see Non-Patent Document 8), and direct conversion techniques to blood stem cells (see Non-Patent Document 9).

  Against this background, we have developed cells capable of forming stratified epithelial tissue that can be directly derived from somatic cells such as skin fibroblasts and adipose-derived mesenchymal cells and that can act as the outer skin of the body. There is a long-awaited desire to provide a source of cells that can be used for the treatment of ulcers, but there have been no reports that cells capable of forming stratified epithelial tissue have been directly converted from somatic cells.

International Publication No. 2007/069666

Kurita M, Ichioka S, Oshima Y, Harii K, “Scand J Plast Reconstr Surg Hand Surg”, 2006, 40 (4), p214-218 Kurita M, Ichioka S, Tanaka Y, Umekawa K, Oshima Y, Ohura N, Kinoshita M, Harii K, “Wound Repair Regenes”, 2009, 17, p312-317 Takahashi K, Yamanaka S, “Cell”, 2006, 25, 126 (4), p663-676 Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S, “Cell”, 2007 Nov 30, 131 (5), p861-72 Davis RL, Weintraub H, Lassar AB, “Cell”, 1987 Dec 24, 51 (6), p987-1000 Vierbuchen T1, Ostermeier A, Pang ZP, Kokubu Y, Sudhof TC, Wernig M, “Nature”, 2010 Feb 25, 463 (7284), p1035-1041 Ieda M, Fu JD, Delgado-Olguin P, Vedantham V, Hayashi Y, Bruneau BG, Srivastava D, “Cell”, 2010 Aug 6, 142 (3), p375-386 Sekiya S1, Suzuki A, “Nature”, 2011 Jun 29, 475 (7356), p390-393

  In view of the above, an object of the present invention is to directly convert, from a somatic cell, a cell capable of forming a stratified epithelial tissue, which can function as an outer coat of the body, in order to solve the above-mentioned problems of the related art. More specifically, the present invention establishes a technique for providing a cell source capable of forming a stratified epithelial tissue that acts as an integument (skin, mucous membrane, corneal epithelium) covering the free surface inside and outside the body. The purpose is to do. Another object of the present invention is to provide various uses of cells capable of forming a stratified epithelial tissue derived from such somatic cells.

  The present inventors have solved dermal fibroblasts as representative somatic cells to solve the above-mentioned problems, and keratinocytes collected from skin as representative cells having the ability to form a stratified epithelial tissue capable of acting as an outer skin. Select and examine the genes expressed in each cell by DNA microarray and microRNA microarray data, and have the same morphology and characteristics as keratinocytes by introducing genes that are specific or relatively strongly expressed in keratinocytes It has been found that cells can be produced. In fact, it was confirmed that the induced fibroblasts thus obtained can be proliferated in a monolayer culture or feeder culture optimized for keratinocyte proliferation, and express a keratinocyte-specific marker. Furthermore, it was confirmed that the induced fibroblasts exhibited self-renewal ability and terminal differentiation under monolayer culture conditions. Furthermore, it was confirmed that when the induced fibroblasts were cultured three-dimensionally using a collagen gel containing skin fibroblasts as a scaffold, a stratified epithelial tissue could be formed. The present invention has been completed by further study based on these findings.

That is, the present invention provides the following aspects of the invention.
Item 1. A method for producing cells having an ability to form stratified epithelium, comprising the step of introducing at least one gene that is relatively strongly expressed in cells having the ability to form stratified epithelium into somatic cells.
Item 2. GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene, ZNF165 gene A step of transfecting one or more genes selected from the ZNF750 gene, ZBED2 gene, IRX2 gene, IRX4 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, KLF4 gene and c-MYC gene into somatic cells. A method for producing a cell capable of forming a stratified epithelial tissue.
Item 3. A method for producing a cell capable of forming a stratified epithelial tissue, comprising the step of introducing (1) the TFAP2A gene or TFAP2C gene, (2) the GRHL family gene, (3) the BNC1 gene, and (4) the MYC family gene into somatic cells. .
Item 4. (1) TFAP2A gene or TFAP2C gene, (2) GRHL family gene, (3) BNC1 gene, (4) MYC family gene, and (5) at least one gene that is relatively strongly expressed in cells having the ability to form stratified epithelial tissue A method for producing a cell having an ability to form a stratified epithelial tissue, comprising the step of introducing the gene into a somatic cell.
Item 5. A method for producing a cell capable of forming a stratified epithelial tissue, which comprises a step of introducing (1) the TFAP2A gene, (2) the GRHL2 gene, (3) the BNC1 gene, and (4) the c-MYC gene into somatic cells.
Item 6. A method for producing a cell having an ability to form a stratified epithelial tissue, comprising a step of introducing a GATA3 gene, a TFAP2A gene, a GRHL2 gene, a TP63 gene, a BNC1 gene, an EHF gene, a ZNF165 gene and a c-MYC gene into a somatic cell.
Item 7. A method for producing cells having an ability to form a stratified epithelium, including the step of introducing OVOL1 gene, TFAP2A gene, GRHL2 gene, BNC1 gene, LASS3 gene, ZBED2 gene, SOX7 gene, SOX9 gene and c-MYC gene into somatic cells. .
Item 8. GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene, ZNF165 gene , Including a step of transfecting somatic cells with a ZNF750 gene, a ZBED2 gene, an IRX2 gene, an IRX4 gene, a SOX7 gene, a SOX9 gene, a FOXQ1 gene, a PPP1R13L gene, a KLF4 gene and a c-MYC gene. Cell production method.
Item 9. Item 9. The method according to any one of Items 1 to 8, wherein the somatic cell is derived from a human.
Item 10. Item 10. The method according to any one of Items 1 to 9, wherein the somatic cell is a skin fibroblast.
Item 11. Item 10. The method according to any one of Items 1 to 9, wherein the somatic cells are adipose tissue-derived stromal cells.
Item 12. Item 10. The method according to any one of Items 1 to 9, wherein the somatic cells are mononuclear cells in peripheral circulating blood.
Item 13. Item 13. A cell capable of forming a stratified epithelial tissue produced by the production method according to any one of Items 1 to 12.
Item 14. Item 14. A cell preparation comprising the cell capable of forming a stratified epithelial tissue according to Item 13.
Item 15. Item 15. The cell preparation according to Item 14, comprising a scaffold material.
Item 16. Item 16. The cell preparation according to Item 15, wherein the scaffold is collagen.
Item 17. Item 17. The cell preparation according to any one of Items 14 to 16, wherein the cell preparation is for regeneration of stratified epithelium, regeneration of skin tissue, regeneration of mucous membrane tissue, or regeneration of corneal tissue.
Item 18. Item 18. The cell preparation according to any one of Items 14 to 17, wherein the cell preparation is a sheet-like epithelial cell sheet.
Item 19. The method according to claim 12, wherein the cell having the ability to form a layered epithelial tissue is administered to a non-human mammal, and the cell having the ability to form a layered epithelial tissue is formed in the body of the mammal by forming a layered epithelial tissue. , A non-human mammal that has formed a stratified epithelial tissue.
Item 20. Item 20. A method for judging the drug effect of a test substance on epithelial tissue, comprising the step of administering the test substance to a non-human mammal according to Item 19 and judging the drug effect of the test substance on epithelial tissue.
Item 21. Item 20. A method for judging the influence of an external factor on epithelial tissue, comprising a step of applying stress such as an anticancer agent or radiation to a non-human mammal according to Item 19 and judging stress on epithelial tissue.
Item 22. Item 9. A composition for preparing a cell having an ability to form a stratified epithelial tissue, comprising the gene introduced into a somatic cell according to any one of Items 1 to 8.
Item 23. Item 22. The cell conditioning composition according to Item 21, wherein the gene is contained in a form that can be introduced into somatic cells.
Item 24. Item 14. An epithelial-like tissue produced by culturing the cell capable of forming a stratified epithelial tissue according to Item 13.
Item 25. An epithelial-like tissue produced by culturing cells capable of forming a stratified epithelial tissue produced by introducing the gene according to any one of Items 1 to 8 into somatic cells taken out from an animal. In contrast, a method for analyzing the efficacy of a test substance on epithelial tissue, comprising the step of administering the test substance and analyzing the efficacy of the test substance on the epithelial-like tissue.
Item 26. An epithelial-like tissue produced by culturing cells capable of forming a stratified epithelial tissue produced by introducing the gene according to any one of Items 1 to 8 into somatic cells taken out from an animal. On the other hand, a method for analyzing the influence of an external factor on epithelial tissue, comprising a step of applying a stress such as an anticancer agent or radiation and analyzing the stress on the epithelial-like tissue.
Item 27. Item 9. A kit for a vector specialized for introducing the gene according to any one of Items 1 to 8 into somatic cells.

  ADVANTAGE OF THE INVENTION According to this invention, the cell which has the ability to form a stratified epithelial tissue having the same characteristics as keratinocytes, gastrointestinal epithelial cells, and corneal epithelial cells can be provided. In addition, such cells having the ability to form a stratified epithelial tissue can be used as a medical means effective for the treatment of skin ulcers such as burns, trauma, pressure ulcers, diabetes, peripheral circulatory insufficiency, gastrointestinal ulcers, and corneal ulcers. Alternatively, a cell preparation can be provided. Further, according to the present invention, not only skin ulcers but also a wide range of diseases that occur in epithelium such as epidermis, gastrointestinal epithelium, and corneal epithelium. By producing cells having the ability to form stratified epithelial tissue from stromal cells, etc., and performing various analyzes on the prepared cells, it is possible to contribute to the elucidation of the disease state or treatment. In particular, cells having the ability to form stratified epithelial tissue prepared from human somatic cells are suitable as a material for drug discovery and drug development in terms of confirming drug efficacy. In addition, the use of cells having the ability to form stratified epithelial tissue having the opposite properties of self-renewal and terminal differentiation can contribute to elucidation of pathological conditions such as cancer originating from epithelial stem cells.

FIG. 4 is a diagram showing data obtained by evaluating the mRNA expression of cultured keratinocytes and cultured fibroblasts by a microarray in a heat map format. The data was obtained from a database (Accession: GDS1505 ID: 1505) provided by NCBI, USA. FIG. 4 is a diagram showing, in a heat map format, data obtained by microarray evaluation of mRNA expression on day 11.5 and day 14.5 of mouse embryos before and after epidermis development. The difference between the gene expressed when the ΔNP63 gene, which is considered to be important for epidermal development, was knocked out, and when it was not, is shown. The figure is cited from Reference 1. FIG. 4 is a diagram showing, in a heat map format, data obtained by microarray evaluation of mRNAs of primary cultured keratinocytes and primary cultured skin fibroblasts established from the same skin specimen. FIG. 4 is a diagram showing data obtained by separating and culturing primary cultured fibroblasts and primary cultured keratinocytes from three skin samples from different sites collected from the same donor and evaluating the amount of expressed microRNA by a microRNA microarray in a heat map format. . It is a schema for a gene transfer test. Lentivirus-directed transfection candidate gene introduction into skin fibroblasts, subculture on mitomycin C-treated 3T3J2 feeder cells, and observation of conversion to cells capable of forming stratified epithelial tissue. Represents. It shows the time course of the gene transfer test. This means that the cells were passaged on the 6th day after gene transfer and continued to be cultured on a feeder in a keratinocyte F medium containing Y27632 which is a Rho kinase inhibitor. FIG. 1 is a diagram of primary cultured keratinocytes cultured on 3T3J2 feeder cells in a keratinocyte F medium containing Y27632 which is a Rho kinase inhibitor. GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene, ZNF165 gene , ZNF750 gene, ZBED2 gene, IRX2 gene, IRX4 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, KLF4 gene, c-MYC gene and KRT14-RFP reporter FIG. It has a clear border with the surrounding feeders and uninduced cells, with flat cells located on the margins of the colonies, showing a keratinocyte colony-like morphology that is stacked towards the center. GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene, ZNF165 gene , ZNF750 gene, ZBED2 gene, IRX2 gene, IRX4 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, KLF4 gene, c-MYC gene and KRT14-RFP reporter FIG. 2 is a view of a colony of the second passage after confirming the above. GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene, ZNF165 gene , ZNF750 gene, ZBED2 gene, IRX2 gene, IRX4 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, KLF4 gene, c-MYC gene and KRT14-RFP reporter FIG. 4 is a diagram of cells obtained by performing cell separation using a label with an anti-Epi-CAM antibody at the second passage after confirming the above (upper panel). It indicates that the cells express Epi-CAM which is a marker of epithelial system. In addition, red fluorescence from the KRT14-RFP reporter was confirmed, indicating that the cells were positive for KRT14 (lower figure). FIG. 6 shows the time-dependent change when 1.8 mM Ca ²⁺ was added to induced fibroblasts on monolayer culture. Left column is without Ca ²⁺ . The right column shows the state with Ca ²⁺ added. Terminal differentiation of induced fibroblasts is initiated by the addition of Ca ²⁺ , indicating that the cell body is expanding flat. At the same time, the expression of KRT14 is decreasing due to the addition of Ca ²⁺ . FIG. 4 shows that induced fibroblasts form stratified keratinized epithelium at the gas-liquid interface by three-dimensional culture. The upper row shows an appearance image during three-dimensional culture. The lower panel shows the histological findings of the stratified epithelium-like tissue composed of collagen gel and induced fibroblasts after three-dimensional culture. By the three-dimensional culture, an epithelial-like tissue in which induced fibroblasts are overlaid on the collagen gel is formed, and a keratinized layer is also observed in the upper layer. Colonies of cells in which insertion of GATA3 gene, TFAP2A gene, GRHL2 gene, TP63 gene, BNC1 gene, EHF gene, ZNF165 gene, c-MYC gene into genomic DNA was confirmed, the left is a high density region, the right is Is a low density region.

1. Method for producing cells having an ability to form stratified epithelial tissue, and use of cells having an ability to form stratified epithelial tissue In the present invention, the term "cells having the ability to form stratified epithelial tissue" refers to the epidermis, the mucous membrane, the cornea, etc. A cell that has the ability to act as a progenitor cell or stem cell of the stratified epithelium that protects the inside of the body from external factors such as mechanical damage and infection at the site (in other words, epithelial stem cell), Somatic cells such as keratinocytes, gastrointestinal epithelial cells, and corneal epithelial cells include cells artificially derived from somatic cells according to the present invention. Cells having the ability to form a stratified epithelial tissue exhibit characteristics of both self-renewal ability and terminal differentiation by adjusting the culture conditions under monolayer culture conditions. Alternatively, cells capable of forming a stratified epithelial tissue have the ability to form stratified epithelial tissue-like structures in three-dimensional culture using a collagen gel containing dermal fibroblasts or the like as a carrier.

  The method for producing cells having an ability to form stratified epithelium according to the present invention is characterized in that somatic cells express relatively strongly in cells having an ability to form stratified epithelium (e.g., keratinocytes, gastrointestinal epithelial cells or corneal epithelial cells). And introducing at least one kind of gene. Hereinafter, a gene to be introduced into a somatic cell is referred to as a “transgene”. The transgene may be a plurality of genes that are relatively strongly expressed in cells having the ability to form stratified epithelial tissue, or in addition to the genes that are relatively strongly expressed in cells having the ability to stratify epithelial tissue, Genes that do not relatively strongly express may be combined.

  Here, the “transgene” includes not only genes encoding proteins but also noncoding RNAs such as microRNAs. In addition, “a gene that is relatively strongly expressed in cells having the ability to form stratified epithelial tissue” refers to a gene whose expression level is higher in cells having the ability to stratify epithelial tissue than mesenchymal cells such as skin fibroblasts. It indicates a gene whose amount is confirmed by a quantitative evaluation method of gene expression such as real-time PCR or microarray. In addition, "a gene which does not relatively strongly express" refers to a gene whose expression level is lower in cells having a stratified epithelial tissue forming ability as compared to mesenchymal cells.

  Genes that are relatively strongly expressed in cells capable of forming stratified epithelial tissue include at least genes encoding the following proteins. TFAP2A gene, TFAP2C gene, GRHL family gene (GRHL1, GRHL2 gene, GRHL3 gene etc.), BNC1 gene, MYC family gene (c-Myc, N-Myc, L-Myc etc.), GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, ESRP2 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, LASS3 gene, EHF gene, ZNF165 gene, ZNF750 gene, ZBED2 gene, IRX4 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene , KLF4 gene. In addition, genes that are relatively strongly expressed in cells capable of forming stratified epithelial tissue include at least the following noncoding RNAs. mir-182, mir-183, mir-96, mir-200b, mir-200a, mir-429, mir-200c, mir-141, mir-203, mir-205, mir-135b, mir-17, mir- 18a, mir-19a, mir-19b-1, mir-20a, mir-92a-1, mir-367.

  In particular, the method for producing cells capable of forming a stratified epithelial tissue according to the present invention comprises: (1) a TFAP2A gene or a TFAP2C gene; (2) at least one gene selected from GRHL family genes; (3) a BNC1 gene; Preferably, the method includes a step of introducing at least one gene selected from MYC family genes into somatic cells. Furthermore, in addition to (1) to (4), (5) at least one gene that is relatively strongly expressed in cells capable of forming a stratified epithelial tissue may be introduced.

  GRHL family genes include the GRHL1, GRHL2, and GRHL3 genes. These GRHL family genes may be used alone or in a combination of two or more. Examples of the Myc family gene include c-Myc, N-Myc, and L-Myc. One of these Myc family genes may be used alone, or two or more thereof may be used in combination. Among these Myc family genes, in the present invention, preferably, the c-Myc gene or the L-Myc gene, and more preferably, the c-Myc gene is used. The c-Myc gene is known as a transcription factor involved in cell differentiation and proliferation (S. Adhikary, M. Elilers, Nat. Rav. Mol. Cell Biol., 6, pp635-645, 2005), Its base sequence is known (see Table 1).

  Further, another method for producing a cell having an ability to form a stratified epithelial tissue according to the present invention includes, as transgenes (direct conversion factors), GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene, ZNF165 gene, ZNF750 gene, ZBED2 gene, IRX2 gene, IRX4 gene, SOX7 gene, SOX9 gene, FOXQ1 gene By introducing all or part of the PPP1R13L gene, the KLF4 gene, and the c-MYC gene into the above-mentioned somatic cells, the somatic cells are induced into cells having the ability to form a stratified epithelial tissue.

  In the present invention, among the genes to be introduced into somatic cells, the TFAP2A gene, GRHL2 gene, BNC1 gene, and c-MYC gene are particularly important, but by introducing the gene in combination with other genes, the cell proliferation ability is improved. Functional characteristics according to the purpose can be imparted, such as an increase, an increase in stratification differentiation ability, and an increase in cell migration ability. For example, GATA3 gene, TFAP2A gene, GRHL2 gene, TP63 gene, BNC1 gene, EHF gene, ZNF165 gene and a production method including a step of transfecting the c-MYC gene into somatic cells, OVOL1 gene, TFAP2A gene, GRHL2 gene, BNC1 gene, LASS3 gene, ZBED2 gene, SOX7 gene, SOX9 gene and c-MYC gene and the production method including the step of transfecting somatic cells, the former can enhance the terminal differentiation ability, the latter can proliferate Can be higher.

  The nucleotide sequence of any of the genes used in the present invention is known (Table 1). In this specification, NCBI is an abbreviation of National Center for Biotechnology Information, and Accession No. in Table 1 is also registered in the database provided by NCBI.

  Many origins of these genes are commonly present in mammals including humans, and those derived from any mammals can be used, but it is desirable to select appropriately according to the origin of the somatic cells to be introduced. . For example, when a human-derived somatic cell is used, the above-mentioned transgene is preferably human. In addition, in addition to the wild-type gene, the above-mentioned transgene has several (for example, 1 to 10, preferably 1 to 6, more preferably 1 to 4, and more preferably 1 to 3) amino acid sequences in its gene product. (Especially 1 or 2) amino acids are substituted, deleted and / or inserted, and encode a mutant gene product having a function equivalent to that of a wild-type gene product. There may be.

  In the present invention, the above-described transgene can be prepared according to a conventional method based on known sequence information. For example, cDNA of the gene of interest can be prepared by extracting RNA from mammalian cells and cloning according to a conventional method.

  In the present invention, the type of “somatic cell” induced by a cell capable of forming a stratified epithelial tissue is not particularly limited, and any type derived from any tissue or site can be used. The somatic cells used in the present invention include, for example, those derived from tissues such as skin, subcutaneous fat, muscle, placenta, bone, cartilage, blood, corneal stroma, and more specifically, dermal fibroblasts , Stromal cells derived from subcutaneous adipose tissue (subcutaneous adipocytes), embryonic fibroblasts, adipocytes, muscle cells, osteoblasts, chondrocytes, circulating blood mononuclear cells, keratocytes in the corneal stroma, and the like. Is done. Among these, skin-derived cells, subcutaneous fat-derived cells, or blood-derived cells are preferable from the viewpoint of minimally invading the living body and producing cells having the ability to form stratified epithelial tissue more efficiently. In particular, skin fibroblasts, subcutaneous adipose tissue-derived stromal cells, and mononuclear cells in circulating blood are preferred. As described above, the material can be selected from various cells, and in particular, skin-derived cells and subcutaneous fat-derived cells, and the availability of easily available cells such as mononuclear cells in the circulating blood can reduce the burden on patients and reduce cell burden. There is also a clinical advantage in terms of stable availability. In addition, as the above-mentioned somatic cells, commercially available products may be used, and somatic cells differentiated from ES cells, mesenchymal stem cells, and the like may also be used.

  The somatic cells may be derived from mammals such as humans, mice, rats, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys, etc., depending on the intended use of the cells having the ability to form stratified epithelial tissue. When used for the purpose of treating humans, elucidating the pathological condition or confirming the efficacy of humans, those derived from humans are preferred. When human-derived somatic cells are used, they may be derived from any of a fetus, an infant, a child, and an adult. When cells having the ability to form a stratified epithelial tissue are used for the purpose of treating humans, elucidating pathological conditions or confirming drug efficacy, it is desirable to use somatic cells collected from patients.

  The above-described transgene can be introduced into somatic cells by a method usually used in transfection of animal cells. Specifically, examples of a method for introducing the transgene into somatic cells include a method using a vector; a calcium phosphate method; a lipofection method; an electroporation method; and a microinjection method. Among these, a method using a vector is preferable from the viewpoint of introduction efficiency. When the transgene is introduced into somatic cells using a vector, viral vectors, non-viral vectors, artificial viruses and the like can be used as vectors. Is preferably used from the viewpoint of safety. In the case where a vector is used, in the case where a plurality of the transgenes are used, each of the transgenes may be incorporated in another vector, or two or more transgenes may be incorporated in one vector. .

  Thus, the somatic cells into which the transgene has been introduced can be induced into cells capable of forming a stratified epithelial tissue. The selection of cells induced to cells having the ability to form stratified epithelial tissue can be performed by isolating keratinocytes, having the ability to proliferate under culture conditions suitable for amplification, and determining whether or not they have characteristics as epithelial cells. It can be carried out. Cells having such an ability to form a stratified epithelial tissue include, specifically, feeder cells (3T3-J2 feeder cells, 3T3 cells, mouse embryonic fibroblasts, human dermal fibroblasts) suitable for isolating and amplifying keratinocytes. Proliferation ability is higher than that of cells that have the ability to form stratified epithelial tissue by culturing cells such as blast cells on mitomycin C or radiation treatment to inactivate the growth ability) and culturing in keratinocyte serum-free medium. Can be selected by continuing the passaging process. It is also effective to add a Rho kinase inhibitor (such as Y27632) that can relatively improve the number of keratinocyte divisions that can be performed on the feeder. In addition, by using a surface antigen (e.g., Epi-CAM) specific to epithelial cells to separate cells using flow cytometry or a magnetic cell separator, the purity of cells capable of forming a layered epithelial tissue can be increased. Is possible. In addition, when somatic cells have previously introduced a reporter gene construct created by linking a drug resistance gene to the promoter of an epithelial cell marker gene (CDH1, Epi-CAM, etc.), the characteristics of epithelial cells are obtained. The cells thus grown can grow in the presence of the drug, and thus can be selected using the growth in the presence of the drug as an index.

  The thus obtained cells having an ability to form a stratified epithelial tissue can be proliferated when cultured in a liquid medium containing a Rho kinase inhibitor on a feeder. Proliferates stably while maintaining tissue forming ability. For culturing cells having the ability to form stratified epithelial tissue, a medium usually used for culturing animal cells can be used. An example of a suitable medium used for culturing cells capable of forming a stratified epithelial tissue includes a serum-free keratinocyte medium (Keratinocyte-SFM, Life technologies) and the like. It is also useful to add cytokines and various pharmacologically active substances, such as bFGF, which accelerate the proliferation of keratinocytes under culture conditions.

  The cells having the ability to form a stratified epithelial tissue obtained in this manner are in vivo on extracellular matrix including mesenchymal cells such as skin fibroblasts and adipose tissue-derived stromal cells, for example, at the wound of skin ulcer. It can form stratified epithelial tissue. Furthermore, by culturing in vitro on a collagen gel containing mesenchymal cells such as skin fibroblasts and adipose tissue-derived stromal cells, a three-dimensional structure of stratified squamous epithelium is formed by forming a gas-liquid interface. Can form an epithelial-like tissue having

  As described above, the cells having the ability to form stratified epithelial tissue obtained by the present invention have a proliferation ability and can regenerate stratified epithelial tissue in and out of a living body. Cell preparation for epithelial tissue regeneration, effective for treating skin ulcers caused by pressure ulcers, diabetes mellitus, peripheral circulatory failure, gastrointestinal ulcers such as nasal cavity, oral cavity, perianal mucosa, etc. (Pharmaceutical composition). The cells having the ability to form a stratified epithelial tissue may be applied alone to the skin, gastrointestinal tract, or corneal disease site.

  When preparing the cell having the ability to form an overlying epithelial tissue as a cell preparation for regenerating epithelial tissue, together with the cell having the ability to form an overlying epithelial tissue, if necessary, containing a pharmaceutically acceptable carrier for dilution. May be. Here, as the pharmaceutically acceptable carrier for dilution, for example, physiological saline, buffer and the like are exemplified. Further, the cell preparation may contain a pharmacologically active component or a component serving as a nutrient source for cells having a capability of forming a stratified epithelial tissue, if necessary.

  The cells having the ability to form a stratified epithelial tissue may be applied to the skin, gastrointestinal tract, or corneal disease site as an epithelial cell sheet formed as a stratified epithelial-like tissue under culture conditions.

  When producing the cell having the ability to form an overlying epithelial tissue as an epithelial cell sheet for regenerating epithelial tissue, together with the cell having the ability to form an overlying epithelial tissue, if necessary, having a pharmacologically active ingredient or an ability to form an overlying epithelial tissue. You may use together with the component used as a nutrient source of a cell.

  The cells having the ability to form a stratified epithelial tissue have a stratified squamous epithelium-like three-dimensional structure using an extracellular matrix such as collagen containing mesenchymal cells such as skin fibroblasts and adipose tissue-derived stromal cells as a scaffold. After preparing epithelial-like tissue, it may be applied to the skin, gastrointestinal tract, or corneal disease site.

  When producing the cell having the ability to form a stratified epithelial tissue as an epithelial-like tissue having a three-dimensional structure for epithelial tissue regeneration, if necessary, together with the cell having the ability to form a stratified epithelial tissue, a pharmacologically active ingredient or stratified epithelium may be used. You may use together with the component used as the nutrient source of the cell which has tissue formation ability. By using such a scaffold material, a stratified epithelium-like structure can be obtained more quickly at a transplant site, and regeneration of epithelial tissue can be further promoted.

  The scaffold material that can be used is not particularly limited as long as it is pharmaceutically acceptable, and is appropriately selected depending on the site of cartilage tissue to be applied. For example, a gel-like material having high biocompatibility (biocompatible ) Materials. Preferred examples of the scaffold material include collagen, fibronectin, hyaluronic acid, and complexes thereof. These scaffold materials may be used alone or in combination of two or more.

  Further, the shape of the scaffold material is not particularly limited, and may be appropriately designed according to the shape of the damaged site of epithelial tissue to which the cell preparation is applied.

  The method for applying the cell preparation to a diseased site in epithelial tissue is appropriately set according to the type of the cell preparation, the site of the epithelial tissue to be applied, and the like. Examples include a method of directly spraying the preparation and a method of suturing and fixing a sheet or a three-dimensional structure according to the skin grafting method.

  In addition, the dosage of the cell preparation applied to the diseased site of the epithelial tissue is effective for the regeneration of the epithelial tissue based on the type of the cell preparation, the site of the epithelial tissue, the degree of the symptom, the age and sex of the patient, etc. May be appropriately set.

  In addition, the cells having the ability to form an overlying epithelial tissue are administered, and a non-human mammal having an epithelial tissue formed from the cells having the ability to form an overlying epithelial tissue evaluates and analyzes the efficacy of the test substance on the epithelial tissue. Can be used as a tool for That is, a test substance is administered to a non-human mammal having an epithelial tissue formed from cells capable of forming the above-mentioned stratified epithelial tissue, and the efficacy of the test substance on the epithelial tissue is determined and analyzed. Evaluate and analyze the efficacy of a substance. Here, the test substance is a substance to be evaluated and analyzed for its effect on epithelial tissue, and specifically includes a candidate substance for a therapeutic drug for epithelial disease. As the non-human mammal, mice, rats, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys, and the like are appropriately selected.

  In addition, in non-human mammals having epithelial tissue formed from cells capable of forming the epithelial epithelium, the effects of external factors, such as anticancer drugs and radiation, that damage the epithelial tissue on the epithelial tissue are examined. Can be used as a model.

  In addition, a three-dimensional structure created by a scaffold such as a collagen gel containing cells having the ability to form a layered epithelial tissue and dermal fibroblasts is used as a tool for evaluating and analyzing the efficacy of a test substance on epithelial tissue. it can. That is, a test substance is administered to a three-dimensional structure created by a scaffold such as a collagen gel containing cells having the ability to form a layered epithelial tissue and dermal fibroblasts, and the drug effect of the test substance on the epithelial tissue is determined and analyzed. By doing so, the efficacy of the test substance on the epithelial tissue can be evaluated and analyzed. Here, the test substance is a substance to be evaluated and analyzed for its effect on epithelial tissue, and specifically includes a candidate substance for a therapeutic drug for epithelial disease. In particular, by using cells with superficial upper formation ability derived from somatic cells that can be collected with relatively low invasiveness, such as peripheral blood circulating mononuclear cells, the drug efficacy for many donors with various genetic backgrounds is widely evaluated, Can be analyzed.

  In addition, the cells having the ability to form stratified epithelial tissue can be used as tools for elucidating and analyzing various epithelial tissue pathologies. It is also useful as a tool for drug discovery and drug development for diseases. For example, a transgene is introduced into a somatic cell taken out of a human body to produce a cell capable of forming a stratified epithelial tissue, and a test substance is administered to an epithelial-like tissue produced by culturing such a cell. It is also possible to evaluate and analyze the drug efficacy of the test substance on the epithelial-like tissue, and to evaluate and analyze the stress on the epithelial-like tissue by applying a stress such as an anticancer agent or radiation. The evaluation and analysis of the drug efficacy or stress may be performed, for example, by comparing a tissue to which a test substance has been administered or a tissue to which stress has been added with a tissue to which no test substance has been administered or given.

  In addition, since the cells having the ability to form stratified epithelial tissue have the characteristics of progenitor cells or stem cells such as self-renewal ability and terminal differentiation, they can form stratified epithelial tissue from somatic cells such as skin fibroblasts that do not have these properties. It can be used as a research tool to elucidate the characteristics of stem cells by analyzing the process of conversion to functional cells.

  Many of the so-called cancers have their origin in the stratified epithelial tissue (squamous cell carcinoma), and the cells capable of forming the stratified epithelial tissue can be used as tools for cancer research.

2. Techniques for Determining Transgene Candidates Hereinafter, the present invention will be described in detail with reference to Examples and the like, but the present invention is not limited thereto.

  Skin fibroblasts were selected as typical somatic cells, and keratinocytes were selected as typical cells having the ability to form stratified epithelial tissue. For the selection of candidate factors that can cause direct conversion, microarray results of expressed genes of cultured human keratinocytes and cultured human dermal fibroblasts were obtained from a database (Accession: GDS1505 ID: 1505) provided by NCBI in the United States (Fig. 1).

  Data obtained by microarray evaluation of mRNA expression at 11.5 days and 14.5 days after embryonic development of the mouse before and after epidermis development were obtained from Reference 1 (FIG. 2).

  To obtain more quantitative data, microarray analysis was performed on primary cultured fibroblasts and primary cultured keratinocytes isolated from the same human skin specimen.

  Isolation and culture of primary cultured fibroblasts were performed by the method described in Reference 2. Specifically, the collected skin specimen was treated overnight at 4 ° C. with 0.25% trypsin, the epidermis was peeled off, explanted on a dish, and a small amount of fibroblast growth medium (FGM) was added. After fibroblasts migrated on the dish, they were passaged when they became subconfluent.

  Separation and culture of primary culture keratinocytes were performed by the method described in Reference 3. Specifically, the collected skin specimen was treated with 0.25% trypsin at 4 ° C. overnight, the dermis was scraped in a serum-free keratinocyte medium (SFKGM), and keratinocytes remaining on the dermis were collected. The culture was continued in SFKGM medium and subcultured when it became subconfluent.

Primary cultured skin fibroblasts, the total RNA was harvested from primary cultures keratinocytes each 10 ⁷ cells, using the Gene ST1.0 microarray (Affimetrix, Inc.), were compared comprehensively mRNA expression (Fig. 3).

For comparison of microRNAs expressing primary cultured fibroblasts and primary cultured keratinocytes, primary cultured fibroblasts and primary cultured keratinocytes were separated and cultured from three skin samples at different sites collected from the same donor, and 10 MicroRNAs were collected from ⁷ cells, and microRNA expression was comprehensively compared using a microRNA microarray, SurePrint G3 Human miRNA microarray (Agilent Technologies) (FIG. 4).

  From the above analysis, at least, TFAP2A gene, TFAP2C gene, GRHL family gene, BNC1 gene, MYC family gene, GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, ESRP2 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene , DNP63A gene, MAPK13 gene, ARNTL2 gene, LASS3 gene, EHF gene, ZNF165 gene, ZNF750 gene, ZBED2 gene, IRX4 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, KLF4 gene, hsa-mir-182, hsa -mir-183, hsa-mir-96, hsa-mir-200b, hsa-mir-200a, hsa-mir-429, hsa-mir-200c, hsa-mir-141, hsa-mir-203, hsa-mir -205, hsa-mir-135b, hsa-mir-17, hsa-mir-18a, hsa-mir-19a, hsa-mir-19b-1, hsa-mir-20a, hsa-mir-92a-1, hsa -mir-367 was confirmed to be a gene that is relatively strongly expressed in keratinocytes.

3. [Example 1] Induction of skin fibroblasts into cells capable of forming stratified epithelial tissue Based on the above analysis results, a lentivirus for selecting 27 transgenes listed in Table 2 and introducing them into somatic cells The vector was prepared. Table 2 shows the transcription factors, microRNAs selected as candidates, and the lentiviral vectors prepared for forced expression and suppression of expression. For the PPP1R13L gene, vectors were prepared for each of the two transcript variants.

  If an appropriate clone exists, a lentivirus vector is purchased from Human Gateway Entry Clone from NBRC (Biotechnology Field of National Institute of Technology and Evaluation) and pLenti7.3 / V5-DEST Gateway Vector (Life Technologies ) Was subcloned according to a conventional method. Others were purchased from manufacturers (System Biosciences, Gene Copoeia, Origene).

Viruses were prepared to introduce the lentiviral vectors of the direct conversion candidate factors in Table 1 into dermal fibroblasts. ViraPower (trademark) HiPerform (trademark) Lentiviral Expression Systems (Life Technologies) was used for virus production. For virus production, 106 293FT cells were seeded on each well of a ^6- well plate, and after overnight, 1 ml of Opti-MEM Reduced Serum Medium with 10% FBS, GlutaMAX (registered trademark) Supplement (Life Technologies) was added to each well. ), The gene was transferred using 600 ng of the construct, 1.2 μg of ViraPower (registered trademark) Lentiviral Packaging Mix, and 7.2 μl of Lipofectamine 2000. After 24 hours, the medium was replaced with 2 ml of 293FT cell growth medium, and after 48 hours, the medium containing the virus solution was recovered. After centrifugation of the virus-containing medium, the supernatant was recovered, and a quarter amount of PEG-it Virus Precipitation Solution (System Biosciences) was added, and the mixture was allowed to stand overnight at 4 ° C. Centrifugation was performed at 1500 × G, the supernatant was discarded, and the precipitate was pipetted with 20 μl of PBS to obtain a virus concentrate. The virus solution collected by the above operation was used as one unit for gene transfer into skin fibroblasts.

  Transdux (registered trademark) (System Biosciences) was used for gene transfer into skin fibroblasts. Twenty-four hours prior to transfection, 48 well plates were seeded with 6000 dermal fibroblasts per well. The medium was changed to 300 μl of a fibroblast growth medium containing a virus solution, and centrifuged at 800 × G for 30 minutes. The medium was replaced again with 300 μl of a fibroblast growth medium containing a new virus solution, and after centrifugation at 800 × G for 30 minutes, the wells were washed with PBS and 300 μl of fibroblast growth medium was added.

 After the gene transfer, the medium was replaced with a fibroblast growth medium on the first day, the second day, and the fourth day after the transfer, and on the sixth day, the cells were placed on 3T3-J2 feeder cells prepared in a mitomycin-treated 6-well plate. Passaged using fibroblast growth medium. On the second day after the passage, the medium was replaced with a keratinocyte F medium containing the Rho-kinase inhibitor Y27632 (Wako), and the medium was replaced every two days thereafter (FIGS. 5 and 6).

The 3T3-J2 feeder cells used as feeders are a cell line provided by J-TEC. Using a 3T3 cell culture medium supplemented with 10% neonatal bovine serum in DMEM medium, maintain the cells in a conventional manner, and treat the cells with mitomycin C 10 μg / ml for 1 hour on the day before use as feeder cells. ^The cells were subcultured at a concentration of ⁵ cells / well and used as a feeder.

  Keratinocyte F medium was prepared according to a conventional method. In this test, 2.5 ml of FBS was added to a mixture of 33.8 ml of F12 medium (Life technologies) and 11.2 ml of high glucose DMEM (Life technologies), and adenine (Sigma) 24 μg / ml, cholera toxin (Wako) 8.4 ng / ml, insulin 5 μg / ml, hydrocortisone (Sigma) 0.4 μg / ml, penicillin (Sigma) 100 U / ml, streptomycin (Sigma) 100 μg / ml, EGF 10 ng / ml Adjusted to ml. Furthermore, in this test, the above keratinocyte F medium was converted to Y-27632 (see Reference 4), indicating that the addition of Rho-kinase inhibitor (Y27632) to the medium on 3T3 feeder cells can extend the life of cultured keratinocytes. Wako) adjusted to 10 μM for use. For comparison, primary cultured keratinocytes were cultured under the same conditions (FIG. 7).

  27 kinds of genes of Example 1 (GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene , LASS3 gene, EHF gene, ZNF165 gene, ZNF750 gene, ZBED2 gene, IRX2 gene, IRX4 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, KLF4 gene and c-MYC gene) Thus, cells exhibiting colonies similar in growth and morphology to keratinocytes were obtained (FIG. 8). In FIG. 8, the colony has a clear border from the surrounding feeder and uninduced cells, flat cells are located at the margin of the colony, and stacked toward the center, and the keratinocytes in FIG. Shows a morphology similar to that of the colony.

  Even if there is no special cell separation, the cells can be propagated in a culture environment on 3T3-J2 feeder cells, which are thought to selectively grow keratinocytes, while maintaining morphology similar to keratinocytes, subculturing and growing. Was possible (FIG. 9). FIG. 9 is a diagram of the colonies of the second passage that were passaged after confirming the induced fibroblast colonies of FIG. From FIG. 9, it can be seen that the induced fibroblasts of Example 1 show a relatively higher proliferative ability under the culture conditions as compared to non-induced fibroblasts that have not been induced into a state similar to keratinocytes. It was confirmed that the operation increased the purity of the induced fibroblasts. In addition, it is possible to obtain highly pure induced fibroblasts in a shorter time by selectively extracting and subculturing keratinocyte-like colonies in the induced fibroblasts of FIG.

  In addition, magnetic cell separation was performed using a magnetically labeled antibody against Epi-CAM, a pan-epithelial cell marker, and it was confirmed that colonies exhibiting a hierarchical growth and morphology more similar to keratinocytes (FIG. 10, upper figure). ). The upper diagram in FIG. 10 is a diagram of cells obtained by confirming the colonies of the induced fibroblasts in FIG. 8 and then performing cell separation using a label with an anti-Epi-CAM antibody at the second passage. Furthermore, it was also confirmed that KRT14 was expressed by the simultaneously introduced KRT14-RFP reporter (pRZ-hKeratin (Cat # SR900CS-1, System Biosciences)) (FIG. 10, lower panel). For magnetic cell separation with magnetically labeled anti-Epi-CAM antibody, CD326 (EpCAM) microbeads, human (Miltenyi Biotech) were used as labeling antibodies, using a manual cell separator and MACS cell separation column (Miltenyi Biotech). I went.

Furthermore, after cell separation using an anti-Epi-CAM antibody, culturing was continued and subcultured, and the expanded induced fibroblasts were subcultured to a monolayer culture condition in serum-free keratinocyte medium. After acclimating the induced fibroblasts to the serum keratinocyte medium, the medium was replaced with a serum-free keratinocyte medium in which the Ca ²⁺ concentration was adjusted to 1.8 mM. It was observed that the cells flattened and expanded over time (FIG. 11). In FIG. 11, the left column shows the state without Ca ²⁺ addition, the right column shows the state with Ca ²⁺ addition, and the red fluorescence state by the KRT14-RFP reporter is also shown at the lapse of 18 hours and 24 hours. Is shown. From the right and left figures, it can be confirmed that terminal differentiation of induced fibroblasts is started by the addition of Ca ²⁺ , and the cell body is flattened. Further, from the figure in the red fluorescence state, it was observed that the expression of KRT14 decreased concurrently. These are the same as the terminal differentiation potential observed when Ca2 ⁺ was added to keratinocytes, indicating that induced dermal fibroblasts under monolayer culture conditions have the same terminal differentiation potential as keratinocytes. I have.

  After cell separation using an anti-Epi-CAM antibody, culturing is continued and subcultured, and cells are cultured on collagen gel containing skin fibroblasts using the expanded induced fibroblasts, and the cells are transferred to the liquid-gas interface. By performing the exposure, a test of the ability to form epithelial tissue was performed using a three-dimensional skin culture model for examining the ability to form epithelial tissue, and it was confirmed that the induced fibroblasts had the ability to form stratified keratinized epithelial tissue under the conditions (FIG. 12). ).

Preparation of the three-dimensional skin culture model was performed using the methods of References 5 and 6. Specifically collagen acidic solution to I-PC 5mg / mL (KOKEN Co.) 3 ml in Hepes ph 7.4, after HaHCO ₃ respectively was added to a final concentration of 10 mM, 10% containing 10 ⁶ fibroblasts Add 4 ml of DMEM medium supplemented with FBS, stir, transfer to a 6 cm dish, and leave in a 37 ° C. incubator for 30 minutes. At the start of gelling, the dish is beaten to float the gel off the dish surface. After that, since the contraction of the gel started, the medium was exchanged every two days, and three-dimensional skin culture with dermal fibroblasts was started on day 7-10.

The collagen gel containing skin fibroblasts thus obtained was placed on a support made of aluminum using a sterilized nylon mesh, and further placed on the collagen gel as shown in the upper diagram of FIG. A glass tube with an inner diameter of 10 mm and an outer diameter of 12 mm is placed. By these operations, the space inside the glass cylinder is separated from the outside of the glass cylinder by the glass cylinder and the collagen gel. The outside of the glass cylinder was filled with a 1: 1 mixed medium of DMEM medium supplemented with 10% FBS and serum-free keratinocyte medium, and 200 μl of the induced fibroblasts that had become subconfluent in one well of a 6-well plate were filled inside the glass cylinder. Sowing with SFKGM. Thereafter, the inner medium was changed daily with serum-free keratinocyte medium, and the outer medium was a one-to-one mixed medium of 10% FBS-added DMEM medium and serum-free keratinocyte medium adjusted to a Ca2 ⁺ concentration of 1.8 mM. The medium was changed once every two days. From day 4, the inner medium was emptied, the level of the outer medium was adjusted to the level of the collagen gel, and the induced fibroblasts were placed in the environment of the liquid-gas interface. Culture was continued until the eyes.

  The lower part of FIG. 12 is a cross-sectional photograph of the induced fibroblasts and the collagen gel after the three-dimensional culture. An epithelial-like tissue layered from the induced fibroblasts is formed on the collagen gel, and the keratinized layer is further formed on the upper layer. Was also formed.

  When a PCR reaction was performed on the genomic DNA of the induced fibroblasts prepared according to the above method using primers specific to the gene derived from the virus vector, the induced fibroblasts contained OVOL1 gene, TFAP2A gene, GRHL2 It was confirmed that nine genes of the gene, BNC1 gene, LASS3 gene, ZBED2 gene, SOX7 gene, SOX9 gene and c-MYC gene were inserted. To confirm the inserted gene derived from the virus vector, a PCR reaction using specific primers shown in Table 3 was performed. For the PCR reaction, using AccuPrime Pfx Supermix (Life technologies), Forward primer and Reverse primer were added to 11.5 μl of AccuPrime Pfx SuperMix reagent per reaction to a final concentration of 200 nM, and 10 ng of Template DNA was added. Thereafter, after a hot start at 95 ° C for 5 minutes in a thermal cycler, a PCR product obtained by repeating a cycle of 95 ° C for 15 seconds, 63 ° C for 30 seconds and 68 ° C for 60 seconds 35 times was subjected to E-Gel (registered trademark) EX. Gel, 1% (INVITROGEN) and electrophoresis using an E-Gel (registered trademark) agarose gel electrophoresis system (INVITROGEN) were used to confirm the presence of the target gene.

  As understood from Example 1, the induction of cells having the ability to form a stratified epithelial tissue involves the GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene, ZNF165 gene, ZNF750 gene, ZBED2 gene, IRX2 gene, IRX4 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, KLF4 gene Not all of the 27 transgenes, c-MYC genes, are required, at least the OVOL1, TFAP2A, GRHL2, BNC1, LASS3, ZBED2, SOX7, SOX9, c-MYC genes By introducing nine types of genes, there is a high possibility that somatic cells can be directly converted into cells capable of forming stratified epithelial tissue. From Example 1, it is meant that by introducing all of these nine genes, cells can be directly converted into cells having the ability to form stratified epithelial tissue, and all nine genes are indispensable for direct conversion. It does not mean that In view of Example 2 described later, it is possible to directly convert cells into cells having the ability to form a stratified epithelial tissue simply by introducing a part of nine genes.

4. [Example 2] Induction of skin fibroblasts into cells capable of forming stratified epithelial tissue From the 27 genes described in Table 2, the remaining 25 genes excluding the IRX2 gene and the IRX4 gene were used. It was introduced into skin fibroblasts as a transgene. Basic experimental conditions and an experimental method are the same as those in the first embodiment. As in Example 1, 25 types of transgenes were introduced into skin fibroblasts, and cultured fibroblasts were obtained. FIG. 13 is a diagram showing colonies of induced fibroblasts prepared according to the above-mentioned method, where the left shows a high-density region and the right shows a low-density region. From FIG. 13, it can be seen from FIG. 13 that the colonies of the induced fibroblasts of Example 2 have clear boundaries with the surrounding feeders and non-induced cells, flat cells are located at the margins of the colonies, and overlap toward the center. And shows a morphology similar to the keratinocyte colony of FIG.

  When a PCR reaction was performed on the genomic DNA of the induced fibroblasts of Example 2 using primers specific to the virus vector-derived gene (Table 3), GATA3 gene, TFAP2A gene, GRHL2 gene, TP63 gene were obtained. It was confirmed that eight genes, the BNC1 gene, the EHF gene, the ZNF165 gene, and the c-MYC gene, were inserted. Cells into which these eight genes have been inserted show proliferation and morphology similar to keratinocytes, but the OVOL1 gene, TFAP2A gene, GRHL2 gene, BNC1 gene, LASS3 gene, ZBED2 gene, SOX7 gene, SOX9 gene, and the OVOL1 gene of Example 1. Compared to cells into which 9 genes of the c-MYC gene were inserted, the cells exhibited a form having higher proliferation ability and lower terminal differentiation ability.

  As understood from Example 2, the induction of cells having the ability to form a stratified epithelial tissue includes the GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene, ZNF165 gene, ZNF750 gene, ZBED2 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, KLF4 gene, c-MYC gene Not all 25 transgenes are required. By introducing at least eight genes, GATA3 gene, TFAP2A gene, GRHL2 gene, TP63 gene, BNC1 gene, EHF gene, ZNF165 gene and c-MYC gene It is highly possible that the cells can be directly converted from somatic cells to cells having the ability to form stratified epithelial tissue. From Example 2, it is meant that by introducing all of these eight genes, cells can be directly converted into cells capable of forming stratified epithelial tissue, and all eight genes are indispensable for direct conversion. It does not mean that

  According to Examples 1 and 2, the cells having the ability to form a stratified epithelial tissue finally derived from somatic cells include the TFAP2A gene, GRHL2 gene, BNC1 gene, c- Four MYC genes were commonly identified. Therefore, by introducing the TFAP2A gene, GRHL2 gene, BNC1 gene, and c-MYC gene, it is highly likely that somatic cells can be directly converted to cells capable of forming stratified epithelial tissue. Further, instead of the TFAP2A gene or in combination with the TFAP2A gene, a TFAP2C gene having a similar function may be introduced, or instead of or in combination with the GRHL2 gene, a GRHL family gene may be introduced. , MYC family genes may be introduced instead of or in combination with the c-MYC gene. Further, in addition to the four genes, at least one gene that is relatively strongly expressed in cells capable of forming a stratified epithelial tissue may be introduced. As can be seen from the difference between the proliferation ability and the terminal differentiation ability between the induced fibroblasts of Example 1 and the induced fibroblasts of Example 2, the cells produced by the combination of the genes to be introduced at the same time. It is possible to regulate characteristics and abilities, for example, proliferation ability, terminal differentiation ability, etc., and the usefulness of adjusting the transgene depending on the purpose of use was confirmed.

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Claims (23)

For somatic cells without the ability to form stratified epithelial tissue,
A method for producing a cell capable of forming a stratified epithelial tissue, comprising a step of introducing a gene into a GATA3 gene, a TFAP2A gene, a GRHL2 gene, a TP63 gene, a BNC1 gene, an EHF gene, a ZNF165 gene, and a c-MYC gene. For somatic cells without the ability to form stratified epithelial tissue,
A method for producing a cell having an ability to form a stratified epithelium, comprising a step of introducing a gene into the OVOL1, TFAP2A, GRHL2, BNC1, LASS3, ZBED2, SOX7, SOX9, and c-MYC genes. For somatic cells without the ability to form stratified epithelial tissue,
GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene, ZNF165 gene , ZNF750 gene, ZBED2 gene, IRX2 gene, IRX4 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, KLF4 gene and c-MYC gene Method. For somatic cells without the ability to form stratified epithelial tissue,
GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene, ZNF165 gene A method for producing a cell having an ability to form a stratified epithelium, which comprises a step of introducing a gene, ZNF750 gene, ZBED2 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, KLF4 gene and c-MYC gene.   The method according to any one of claims 1 to 4, wherein the somatic cell is derived from a human.   The method according to any one of claims 1 to 5, wherein the somatic cells are dermal fibroblasts.   The method according to any one of claims 1 to 5, wherein the somatic cells are adipose tissue-derived stromal cells.   The method according to any one of claims 1 to 5, wherein the somatic cells are mononuclear cells in peripheral circulating blood.   A cell having an ability to form a stratified epithelial tissue, which is produced by the production method according to claim 1.   A cell preparation comprising the cell capable of forming a stratified epithelial tissue according to claim 9.   11. The cell preparation according to claim 10, comprising a scaffold material.   The cell preparation according to claim 11, wherein the scaffold is collagen.   The cell preparation according to any one of claims 10 to 12, wherein the cell preparation is for regeneration of stratified epithelium, regeneration of skin tissue, regeneration of mucosal tissue, or regeneration of corneal tissue.   The cell preparation according to any one of claims 10 to 13, wherein the cell preparation is a sheet-like epithelial cell sheet.   A cell having the ability to form stratified epithelial tissue according to claim 9, which is administered to a non-human mammal to form stratified epithelial tissue from the cell capable of forming stratified epithelial tissue in the body of the mammal. A non-human mammal having formed a stratified epithelial tissue.   A method for determining the efficacy of a test substance on epithelial tissue, comprising the step of administering the test substance to the non-human mammal according to claim 15 and determining the efficacy of the test substance on epithelial tissue.   A method for determining the influence of an external factor on epithelial tissue, comprising a step of applying a stress such as an anticancer agent or radiation to the non-human mammal according to claim 15 and determining a stress on epithelial tissue. (A) a combination of eight genes consisting of GATA3 gene, TFAP2A gene, GRHL2 gene, TP63 gene, BNC1 gene, EHF gene, ZNF165 gene and c-MYC gene,
(B) a combination of nine genes consisting of OVOL1, TFAP2A, GRHL2, BNC1, LASS3, ZBED2, SOX7, SOX9 and c-MYC genes;
(C) GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene , ZNF165 gene, ZNF750 gene, ZBED2 gene, IRX2 gene, IRX4 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, KLF4 gene and a combination of 27 genes consisting of c-MYC gene, or
(D) GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene , ZNF165 gene, ZNF750 gene, ZBED2 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, combination of 25 genes consisting of KLF4 gene and c-MYC gene,
A composition for preparing cells having the ability to form a stratified epithelium, comprising:   19. The composition for cell preparation according to claim 18, wherein the gene is contained in a form that can be introduced into somatic cells.   An epithelial cell tissue differentiated from the cell capable of forming a stratified epithelial tissue according to claim 9. Somatic cells taken from animals
(A) a combination of eight genes consisting of GATA3 gene, TFAP2A gene, GRHL2 gene, TP63 gene, BNC1 gene, EHF gene, ZNF165 gene and c-MYC gene,
(B) a combination of nine genes consisting of OVOL1, TFAP2A, GRHL2, BNC1, LASS3, ZBED2, SOX7, SOX9 and c-MYC genes;
(C) GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene , ZNF165 gene, ZNF750 gene, ZBED2 gene, IRX2 gene, IRX4 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, KLF4 gene and a combination of 27 genes consisting of c-MYC gene, or
(D) GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene , ZNF165 gene, ZNF750 gene, ZBED2 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, combination of 25 genes consisting of KLF4 gene and c-MYC gene,
Administering a test substance to an epithelial cell tissue differentiated from cells capable of forming a stratified epithelial tissue produced by introducing a gene, and determining the efficacy of the test substance on the epithelial tissue. Method of analyzing the medicinal properties of Somatic cells taken from animals
(A) a combination of eight genes consisting of GATA3 gene, TFAP2A gene, GRHL2 gene, TP63 gene, BNC1 gene, EHF gene, ZNF165 gene and c-MYC gene,
(B) a combination of nine genes consisting of OVOL1, TFAP2A, GRHL2, BNC1, LASS3, ZBED2, SOX7, SOX9 and c-MYC genes;
(C) GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene , ZNF165 gene, ZNF750 gene, ZBED2 gene, IRX2 gene, IRX4 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, KLF4 gene and a combination of 27 genes consisting of c-MYC gene, or
(D) GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene , ZNF165 gene, ZNF750 gene, ZBED2 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, combination of 25 genes consisting of KLF4 gene and c-MYC gene,
Applying a stress, such as an anticancer agent or radiation, to epithelial cell tissue differentiated from cells having the ability to form stratified epithelial tissue produced by introducing the gene, and determining the stress on the epithelial tissue. How to analyze the impact of external factors on the organization. (A) a combination of eight genes consisting of GATA3 gene, TFAP2A gene, GRHL2 gene, TP63 gene, BNC1 gene, EHF gene, ZNF165 gene and c-MYC gene,
(B) a combination of nine genes consisting of OVOL1, TFAP2A, GRHL2, BNC1, LASS3, ZBED2, SOX7, SOX9 and c-MYC genes;
(C) GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene , ZNF165 gene, ZNF750 gene, ZBED2 gene, IRX2 gene, IRX4 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, KLF4 gene and a combination of 27 genes consisting of c-MYC gene, or
(D) GATA3 gene, OVOL1 gene, OVOL2 gene, ESRP1 gene, TFAP2A gene, ID1 gene, GRHL1 gene, GRHL2 gene, GRHL3 gene, TP63 gene, DNP63A gene, MAPK13 gene, ARNTL2 gene, BNC1 gene, LASS3 gene, EHF gene , ZNF165 gene, ZNF750 gene, ZBED2 gene, SOX7 gene, SOX9 gene, FOXQ1 gene, PPP1R13L gene, combination of 25 genes consisting of KLF4 gene and c-MYC gene,
A kit comprising a vector specialized for introducing a gene into a somatic cell.

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